1. Field of the Invention
The present invention relates to variants having chymotrypsin-like activity, nucleic acids encoding the variants, methods of producing the variants, and methods for using the variants.
2. Description of the Related Art
Proteolytic enzymes have widespread commercial application and have been successfully implemented in different industries such as the detergent, leather, chemical, agricultural, pharmaceutical, food, and dairy industries. Chymotrypsin and trypsin are two such proteolytic enzymes available from mammalian sources. Chymotrypsin preferentially cleaves at the C-terminal side of peptide bonds of the L-isomers of tyrosine, phenylalanine, and tryptophan. Trypsin preferentially cleaves at the C-terminal side of peptide bonds of the L-isomers of lysine and arginine. Mammalian trypsin and chymotrypsin are synthesized as precursors known as trypsinogen and chymotrypsinogen, respectively, having both an amino-terminal signal peptide to direct secretion as well as a propeptide that silences enzyme activity until it is proteolytically removed with concomitant activation of the enzyme. Cleavage of the propeptide requires a highly specific serine endoprotease activity.
Four chymotrypsin-like serine proteases have been identified from Streptomyces griseus, namely SGT, SGPA, SGPB, and SGPE (Awad et al., 1972, Journal of Biological Chemistry 247: 4144-4154; Yoshida et al., 1988, J. Biochem. (Tokyo) 104: 451-456). The gene sequences of these chymotrypsin-like serine proteases have also been disclosed (Henderson et al., 1987, Journal of Bacteriology 169: 3778-3784; Sidhu et al., 1993, Biochem. Cell. Biol. 71: 454-461; and Kim et al., 1991, Biochem. Biophys. Res. Commun. 181: 707-713). Sachdev et al., 1994, Journal of Biological Chemistry 269: 20167-20171, disclose a Streptomyces griseus chymotrypsin-like serine protease designated SCPC and the gene encoding the protease. Screen and St. Leger, 2000, Journal of Biological Chemistry 275: 6689-6694, disclose a chymotrypsin-like enzyme from the deuteromycete Metarhizium anisopliae. 
Hedstrom et al., 1992, Science 255: 1249-1253 disclose the protein engineering of a mammalian trypsin gene to code for a polypeptide with a functional chymotrypsin substrate profile by site-directed mutagenesis of the S1 binding site and surface loops of the binding pocket of trypsin with analogous residues of chymotrypsin.
While chymotrypsin is obtainable from mammalian sources and chymotrypsin-like enzymes are available from a few microbial sources, there is a need in the art for new sources of chymotrypsin-like enzymes to provide alternative sources to establish new enzymatic processes and to provide improved cost and performance.
The object of the present invention is to provide protein engineered microbial polypeptides having chymotrypsin-like activity from microbial trypsin-like enzymes.